Monocyte-derived macrophages, the precursors of the lipid-laden foam cells in fatty-streak lesions, are critically important in the initiation of atherosclerosis. During earlier funding periods, we discovered and cloned CCR2, the chemokine receptor that regulates monocyte migration to MCP-1, and showed that CCR2-/- mice are protected in murine models of atherosclerosis. Recent studies during the past funding period revealed considerable heterogeneity among circulating blood monocytes and a pivotal role for CCR2 in monocyte egress from the bone marrow to the blood. Building on this scientific progress, we propose three interrelated specific aims to define the role of CCR2 and its chemokine ligands both in the the migration of monocytes from the bone marrow to the blood and thence to atherosclerotic lesions, and in the activation of macrophages within existing lesions. Our working model is that CCR2 is the key receptor in mediating the migration of a subpopulation of "inflammatory" monocytes from the vascular niche of the bone marrow to developing atherosclerotic lesions. Specific Aim 1 will define the mechanism(s) for CCR2-dependent monocyte egress from the bone marrow. Using novel CCR2-RFP knock-in mice and MCP-1/MCP-3 lac-Z reporter mice, we will test the hypothesis that CCR2 mediates selective movement of monocytes and monocyte progenitor cells from the bone marrow osteoblastic niche to the vascular niche for release into the peripheral blood. Specific Aim 2 will identify the monocyte population(s) recruited to atherosclerotic lesions and the chemokine receptors that direct this trafficking. Using CCR2-RFP mice and adoptive transfer of subpopulations of bone marrow monocytes, we will test the hypothesis that CCR2-expressing cells are selectively recruited to developing atherosclerotic lesions. Specific Aim 3 will determine if pharmacological blockade of CCR2 facilitates lesion regression or reduces the activity of lesional macrophage metalloproteinases or cathepsins. Using novel human CCR2 knock-in mice and near-infrared imaging of protease-specific fluorescent "beacons," we will determine if potent CCR2 antagonists reduce the inflammatory potential of macrophages within lesions. Completion of these studies will deepen our understanding of the role of chemokines in monocyte homeostasis and the importance of macrophage activation in atherosclerotic plaque instability.